U.S. patent application number 12/896246 was filed with the patent office on 2011-12-15 for synthesis and biological evaluation of 2',5'-dimethoxychalcone derivatives as microtubule-targeted anticancer agents.
This patent application is currently assigned to KAOHSIUNG MEDICAL UNIVERSITY. Invention is credited to Jan-Gowth Chang, Tzyh-Chyuan Hoar, A-Mei Huang, Chun-Nan Lin, Yeong-Shiau Pu, Huang-Yao Tu, Shyh-Chyun Yang.
Application Number | 20110306775 12/896246 |
Document ID | / |
Family ID | 45096745 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306775 |
Kind Code |
A1 |
Lin; Chun-Nan ; et
al. |
December 15, 2011 |
SYNTHESIS AND BIOLOGICAL EVALUATION OF 2',5'-DIMETHOXYCHALCONE
DERIVATIVES AS MICROTUBULE-TARGETED ANTICANCER AGENTS
Abstract
Disclosed are a serious of 2',5'-dimethoxychalcone derivatives
for treating cancer, wherein 2,5-dimethoxyacetophenone and methyl
4-formylbenzoate are condensed to form
4-carboxyl-2',5'-dimethoxychalcone (compound 1), which is further
reacted with alkyl halides or amines to synthesize the chalcone
derivatives of compounds 2-17. In addition,
2,5-dimethoxyacetophenone is reacted with
5-formyl-2-thiophenecarboxylic acid to form compound 18
(3-(3-thiophene)carboxyl-1-(2,5-dimethoxyphenyl)prop-2-en-1-one).
The synthesized 2',5'-dimethoxychalcone derivatives can be acted as
microtubule-targeted tubulin-polymerizing agents.
Inventors: |
Lin; Chun-Nan; (Kaohsiung
City, TW) ; Tu; Huang-Yao; (Kaohsiung City, TW)
; Huang; A-Mei; (Kaohsiung City, TW) ; Hoar;
Tzyh-Chyuan; (Kaohsiung City, TW) ; Yang;
Shyh-Chyun; (Kaohsiung City, TW) ; Pu;
Yeong-Shiau; (Taipei City, TW) ; Chang;
Jan-Gowth; (Kaohsiung City, TW) |
Assignee: |
KAOHSIUNG MEDICAL
UNIVERSITY
Kaohsiung City
TW
|
Family ID: |
45096745 |
Appl. No.: |
12/896246 |
Filed: |
October 1, 2010 |
Current U.S.
Class: |
548/539 ; 549/71;
560/53; 562/463; 564/169 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 207/06 20130101; C07C 59/90 20130101; C07D 333/40
20130101 |
Class at
Publication: |
548/539 ;
562/463; 549/71; 560/53; 564/169 |
International
Class: |
C07D 207/12 20060101
C07D207/12; C07C 233/00 20060101 C07C233/00; C07C 69/76 20060101
C07C069/76; C07C 59/90 20060101 C07C059/90; C07D 333/38 20060101
C07D333/38 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2010 |
TW |
099118967 |
Claims
1. A pharmaceutical composition for treating a cancer comprising a
chalcone compound represented by formula I: ##STR00014## wherein R1
and R2 have one of the following two situations, that (i) R1 is a
first substituted group being one selected from a group consisting
of a hydroxy group, a C.sub.1-C.sub.6 alkoxy group, an aromatic
alkoxy group, a heterocyclic alkoxy group, an alkenyloxy group, an
alkylamide group, a C.sub.3-C.sub.6 cycloalkylamide group, a
heterocyclic amide group and an
--N(C.sub.mH.sub.2m+1)--C.sub.nH.sub.2nOH group with
1.ltoreq.m.ltoreq.6 and when R2 is an oxygen, and (ii) R1 is a
cyclopropylamide group when R2 is a cyclopropylimino group.
2. The pharmaceutical composition according to claim 1, wherein the
alkenyloxy group is one of an --OCH.sub.2CH.dbd.CHC.sub.rH.sub.2r+1
group with 1.ltoreq.r.ltoreq.6 and an
--OCH.sub.2CH.dbd.C(C.sub.pH.sub.2p+1)--C.sub.qH.sub.2q+1 group
with 1.ltoreq.p.ltoreq.6 and 1.ltoreq.q.ltoreq.6.
3. The pharmaceutical composition according to claim 1, wherein the
alkylamide group is represented as an --NR--R' group, and each of R
and R' is a second substituted group being one selected from a
group consisting of a hydrogen, a C.sub.1-C.sub.6 saturated
hydrocarbon group and a C.sub.1-C.sub.6 unsaturated hydrocarbon
group.
4. A pharmaceutical composition for treating a cancer comprising a
chalcone compound represented by formula II: ##STR00015##
5. The pharmaceutical composition according to claim 4, wherein the
chalcone compound is synthesized by reacting a
2,5-dimethoxyacetophenone with a 5-formyl-2-thiophenecarboxylic
acid.
6. A preparation method of a pharmaceutical composition for
treating a cancer, the method comprising a step of: (a) reacting
2,5-dimethoxyacetophenone with a methyl 4-formylbenzoate to obtain
a 4-carboxyl-2',5'-dimethoxychalcone.
7. The method according to claim 6, wherein the step (a) is
performed in an alkaline solution and then is neutralized in a
first acidic solution.
8. The method according to claim 7, wherein the alkaline solution
is a potassium hydroxide solution, and the first acidic solution is
a hydrochloride solution.
9. The method according to claim 6 further comprising a step of
(b1) reacting the 4-carboxyl-2',5'-dimethoxychalcone with an alkyl
halide to obtain a first compound represented by formula I:
##STR00016## wherein R1 is a first substituted group being one
selected from a group consisting of a hydroxy group, a
C.sub.1-C.sub.6 alkoxy group, an aromatic alkoxy group, a
heterocyclic alkoxy group and an alkenyloxy group when R2 is an
oxygen.
10. The method according to claim 9, wherein the alkenyloxy group
is one of an --OCH.sub.2CH.dbd.CHC.sub.rH.sub.2r+1 group with
1.ltoreq.r.ltoreq.6 and an
--OCH.sub.2CH.dbd.C(C.sub.pH.sub.2p+1)--C.sub.qH.sub.2q+1 group
with 1.ltoreq.p.ltoreq.6 and 1.ltoreq.q.ltoreq.6.
11. The method according to claim 9, wherein the step (b1) further
comprises: (b11) reacting the 4-carboxyl-2',5'-dimethoxychalcone
and the alkyl halide with a potassium carbonate to obtain a first
reaction mixture; (b12) concentrating the first reaction mixture;
(b13) neutralizing the first reaction mixture with a second acidic
solution; and (b 14) extracting the first reaction mixture with a
first dichloromethane to obtain the first compound.
12. The method according to claim 11, wherein the second acidic
solution is a hydrochloride solution.
13. The method according to claim 6 further comprising a step of:
(b2) reacting 4-carboxyl-2',5'-dimethoxychalcone with an amine to
obtain a second compound represented by formula I: ##STR00017##
wherein R1 is a second substituted group being one selected from a
group consisting of an alkylamide group, a C.sub.3-C.sub.6
cycloalkylamide group, a heterocyclic amide group and a
--N(C.sub.mH.sub.2m+1)--C.sub.nH.sub.2nOH group with
1.ltoreq.m.ltoreq.6 and 1.ltoreq.n.ltoreq.6 when R2 is an
oxygen.
14. The method according to claim 13, wherein the alkylamide group
is an --NR--R' group, and each of R and R' is a third substituted
group being one selected from a group consisting of a hydrogen, a
C.sub.1-C.sub.6 saturated hydrocarbon group and a C.sub.1-C.sub.6
unsaturated hydrocarbon group.
15. The method according to claim 13, wherein the step (b2) further
comprises: (b21) dissolving 4-carboxyl-2',5'-dimethoxychalcone,
hydroxybenzotriazole (HOBt) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl)
in a dichloromethane to form a second mixture; (b22) reacting the
second mixture with the amine to obtain a second reaction mixture;
(b23) concentrating the second reaction mixture; and (b24)
crystallizing the second reaction mixture with an ethyl acetate to
obtain the second compound.
16. A preparation method of a pharmaceutical composition for
treating a cancer, comprising a step of reacting a
2,5-dimethoxyacetophenone with a 5-formyl-2-thiophenecarboxylic
acid to obtain a compound represented by formula II:
##STR00018##
17. The preparation method according to claim 16, wherein the
2,5-dimethoxyacetophenone and the 5-formyl-2-thiophenecarboxylic
acid are reacted in an alkaline solution and then neutralized in an
acidic solution.
18. The preparation method according to claim 17, wherein the
alkaline solution is a potassium hydroxide solution, and the acidic
solution is a hydrochloride solution.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an anticancer agent and its
preparation method. In particular, the present invention relates to
a microtubule-targeted anticancer agent and the preparation method
thereof.
BACKGROUND OF THE INVENTION
[0002] Microtubules mainly compose of tubulins, and microtubules
plays an important role in a variety of cellular process including
mitosis and cell division. Various anti-mitotic agents interfering
with the natural dynamics of tubulin, polymerization, and
depolymerization inhibit cancer cell growth (Kim et al., 2004).
[0003] For instance, paclitaxel stabilizes microtubules by
preventing the depolymerization of tubulin while the vinca
alkaloids and colchicine inhibit the polymerization of tubulin.
Disruption of tubulin dynamics leads to cell cycle arrest in the
G2/M phase and induction of apoptosis (Kim et al., 2004). At
present, a major problem of anti-mitotic agent (such as taxanes and
vinca alkaloids) in clinical application is the development of drug
resistance. Therefore, it needs to find and develop effective
tubulin inhibitors for treating multidrug-resistant (MDR)
tumors.
[0004] It is therefore attempted by the applicant to deal with the
above situation encountered in the prior art.
SUMMARY OF THE INVENTION
[0005] For developing new anti-mitotic agents, the novel chalcones
are synthesized in the present invention. Chalcones are ketones
having aromatic rings and have been characterized with multiple
biological activities including anti-inflammatory, anti-malaria,
anti-protozoal, anti-bacterial, nitric oxide inhibitory, tyrosinase
inhibitory, cytotoxic, anti-leishmanial, and anti-oxidant
activities (Mukherjee et al., 2001). The C-4 of ring B of the newly
synthesized 2',5'-dialkoxylchalcones is conjugated with carbonyl
group (C.dbd.O) or carbamoyl group (--R--CONH.sub.2). The
experiments indicate that the compounds synthesized in the present
invention have effective cytotoxicity on the urinary system-related
cancer cells and show less cytotoxicity on normal cells. However,
the compounds of the present invention also can be applied in
cytotoxicity on other cancer cells.
[0006] The first aspect of the present invention is to provide a
pharmaceutical composition for treating a cancer including a
chalcone compound represented by formula I:
##STR00001##
R1 and R2 have one of the following two situations, that (i) R1 is
a first substituted group being one selected from a group
consisting of a hydroxy group, a C.sub.1-C.sub.6 alkoxy group, an
aromatic alkoxy group, a heterocyclic alkoxy group, an alkenyloxy
group, an alkylamide group, a C.sub.3-C.sub.6 cycloalkylamide
group, a heterocyclic amide group and an
--N(C.sub.mH.sub.2m+1)--C.sub.nH.sub.2nOH group with
1.ltoreq.m.ltoreq.6 and 1.ltoreq.n.ltoreq.6 when R2 is an oxygen,
and (ii) R1 is a cyclopropylamide group when R2 is a
cyclopropylimino group.
[0007] Preferably, the alkenyloxy group is an
--OCH.sub.2CH.dbd.CHC.sub.rH.sub.2r+1 group with
1.ltoreq.r.ltoreq.6 or an
--OCH.sub.2CH.dbd.C(C.sub.pH.sub.2p+1)--C.sub.qH.sub.2q+1 group
with 1.ltoreq.p.ltoreq.6 and 1.ltoreq.q.ltoreq.6.
[0008] Preferably, the alkylamide group is represented as an
--NR--R' group, and each of R and R' is a second substituted group
being one selected from a group consisting of a hydrogen, a
C.sub.1-C.sub.6 saturated hydrocarbon group and a C.sub.1-C.sub.6
unsaturated hydrocarbon group.
[0009] The second aspect of the present invention is to provide a
pharmaceutical composition for treating a cancer including a
chalcone compound represented by formula II:
##STR00002##
The chalcone compound is synthesized by reacting a
2,5-dimethoxyacetophenone with a 5-formyl-2-thiophenecarboxylic
acid.
[0010] The third aspect of the present invention is to provide a
preparation method of a pharmaceutical composition for treating a
cancer, and the method includes a step of: (a) reacting
2,5-dimethoxyacetophenone with a methyl 4-formylbenzoate to obtain
a 4-carboxyl-2',5'-dimethoxychalcone.
[0011] Preferably, the step (a) is performed in an alkaline
solution and then is neutralized in a first acidic solution. The
alkaline solution is a potassium hydroxide solution, and the first
acidic solution is a hydrochloride solution.
[0012] Preferably, the method further includes a step of: (b1)
reacting the 4-carboxyl-2',5'-dimethoxychalcone with an alkyl
halide to obtain a first compound represented by the aforementioned
formula I, wherein R1 is a first substituted group being one
selected from a group consisting of a hydroxy group, a
C.sub.1-C.sub.6 alkoxy group, an aromatic alkoxy group, a
heterocyclic alkoxy group and an alkenyloxy group when R2 is an
oxygen.
[0013] Preferably, the alkenyloxy group is an
--OCH.sub.2CH.dbd.CHC.sub.rH.sub.2r+1 group with
1.ltoreq.r.ltoreq.6 or an
--OCH.sub.2CH.dbd.C(C.sub.pH.sub.2p+1)--C.sub.qH.sub.2q+1 group
with 1.ltoreq.p.ltoreq.6 and 1.ltoreq.q.ltoreq.6.
[0014] Preferably, the step (b1) further includes: (b11) reacting
the 4-carboxyl-2',5'-dimethoxychalcone and the alkyl halide with a
potassium carbonate to obtain a first reaction mixture; (b12)
concentrating the first reaction mixture; (b13) neutralizing the
first reaction mixture with a second acidic solution; and (b14)
extracting the first reaction mixture with a first dichloromethane
to obtain the first compound. The second acidic solution is a
hydrochloride solution.
[0015] Preferably, the method further includes a step of: (b2)
reacting 4-carboxyl-2',5'-dimethoxychalcone with an amine to obtain
a second compound represented by the aforementioned formula I,
wherein R1 is a second substituted group being one selected from a
group consisting of an alkylamide group, a C.sub.3-C.sub.6
cycloalkylamide group, a heterocyclic amide group and a
--N(C.sub.mH.sub.2m+1)--C.sub.2H.sub.2nOH group with
1.ltoreq.m.ltoreq.6 and 1.ltoreq.n.ltoreq.6 when R2 is an oxygen.
The alkylamide group is an --NR--R' group, and each of R and R' is
a third substituted group being one selected from a group
consisting of a hydrogen, a C.sub.1-C.sub.6 saturated hydrocarbon
group and a C.sub.1-C.sub.6 unsaturated hydrocarbon group.
[0016] Preferably, the step (b2) further includes: (b21) dissolving
4-carboxyl-2',5'-dimethoxychalcone, hydroxybenzotriazole (HOBt) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl)
in a dichloromethane to form a second mixture; (b22) reacting the
second mixture with the amine to obtain a second reaction mixture;
(b23) concentrating the second reaction mixture; and (b24)
crystallizing the second reaction mixture with an ethyl acetate to
obtain the second compound.
[0017] The fourth aspect of the present invention is to provide a
preparation method of a pharmaceutical composition for treating a
cancer, and the method includes a step of reacting a
2,5-dimethoxyacetophenone with a 5-formyl-2-thiophenecarboxylic
acid to obtain a compound represented by the aforementioned formula
II.
[0018] Preferably, the 2,5-dimethoxyacetophenone and the
5-formyl-2-thiophenecarboxylic acid are reacted in an alkaline
solution and then neutralized in an acidic solution. The alkaline
solution is a potassium hydroxide solution, and the acidic solution
is a hydrochloride solution.
[0019] The above objectives and advantages of the present invention
will become more readily apparent to those ordinarily skilled in
the art after reviewing the following detailed descriptions and
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 depicts the cytotoxicities of compound 15 against
NTUB1, PC3, A549 and SV-HUC1 cells.
[0021] FIG. 2 depicts the cell cycle distribution of NTUB1 cells
for 24 hours with compounds 11 and 15 in different
concentrations.
[0022] FIG. 3 depicts the cell cycle distribution of PC3 cells
treated with compounds 11 and 15 in different concentrations.
[0023] FIG. 4 depicts immunoblotting of .alpha.-tubulin in NTU
cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0024] The present invention will now be described more
specifically with reference to the following Embodiments. It is to
be noted that the following descriptions of preferred Embodiments
of this invention are presented herein for purpose of illustration
and description only; it is not intended to be exhaustive or to be
limited to the precise form disclosed.
EXPERIMENTAL METHODS
[0025] I. Preparation of compound 1 to 18:
[0026] 1. Preparation of compound 1
(4-carboxyl-2',5'-dimethoxychalcone)
[0027] 2,5-Dimethoxyacetophenone (450.0 mg, 2.5 mmol) and methyl
4-formylbenzoate (410.4 mg, 2.5 mmol) were dissolved in methanol
(MeOH, 50 mL), and added 8% potassium hydroxide (KOH) in H.sub.2O
(50 mL). The reaction mixture was stirred at room temperature for
24 hours and neutralized with 10% hydrochloride solution (HCl, 100
mL) to form yellow precipitate. The yellow precipitate was filtered
and washed with appropriate amount of water. The crude product was
purified by chromatography using ethyl acetate (EtOAc)/n-hexane
(2:1), and crystallized by EtOAc/n-hexane (1:4) to afford compound
1 (4-carboxyl-2',5'-dimethoxychalcone, 491.6 mg, 63.0%) as a yellow
solid. The reaction formula of compound 1 was shown as follows.
##STR00003##
[0028] The properties of compound 1 were listed as follows. IR
(KBr): 1681, 1597 cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 3.82
(3H, s, OCH.sub.3), 3.89 (3H, s, OCH.sub.3), 6.96 (1H, d, J=8.8 Hz,
H-3'), 7.06 (1H, dd, J=9.0, 3.2 Hz, H-4'), 7.23 (1H, d, J=3.2 Hz,
H-6'), 7.55 (1H, d, J=16.0 Hz, H-.alpha.), 7.68 (1H, d, J=16.0 Hz,
H-.beta.), 7.69 (2H, dd, J=8.8, 1.6 Hz, H-2 and 6), 8.13 (2H, dd,
J=8.8, 1.6 Hz, H-3 and 5). .sup.13C NMR (CDCl.sub.3): .delta. 55.9
(OCH.sub.3), 56.5 (OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'), 119.8
(C-1' and 4'), 119.8 (C-.alpha.), 128.3 (C-2 and 6), 129.3 (C-3 and
5), 130.2 (C-4), 140.4 (C-1), 141.0 (C-.beta.), 152.8 (C-5'), 153.7
(C-2'), 171.0 (--COOH), 191.8 (C.dbd.O). ESIMS: m/z 313
[M+H].sup.+. Anal. Calcd for C.sub.18H.sub.16O.sub.5: C, 69.22; H,
5.16. Found: C, 69.10; H, 5.18.
[0029] 2. Procedure A for synthesis of C-4 ester compounds 2 to
7
[0030] To a solution of compound 1 (60 mg, 0.19 mmol) in acetone
was added potassium carbonate (K.sub.2CO.sub.3, 52.5 mg, 0.38 mmol)
and alkyl halide (0.38 mmol). The reaction mixture was stirred at
room temperature for preparing compounds 2 to 4 or refluxed for
preparing compounds 5 to 7 overnight. The mixture was concentrated
to dryness under reduced pressure, neutralized with 10% HCl
solution, and extracted with dichloromethane (CH.sub.2Cl.sub.2).
The organic phase was dried over an hydrous sodium sulfate
(Na.sub.2SO.sub.4), filtered, and concentrated in vacuo to give the
crude product. The crude product was purified by chromatography
with EtOAc/n-hexane, and crystallized with acetone/n-hexane (1:4)
to afford purified products. Compounds 2 to 7 have the structural
formula (Formula I) shown as follows, wherein R1 and R2 substituted
groups are depicted in Table 1.
##STR00004##
[0031] The detailed descriptions for compounds 2 to 7 are provided
as follows.
[0032] 3. Preparation of compound 2
(4-methoxycarbonyl-2',5'-dimethoxychalcone)
[0033] To a solution of compound 1 (60 mg, 0.19 mmol) in acetone
was added K.sub.2CO.sub.3 (52.5 mg, 0.38 mmol) and methyl iodide
(0.38 mmol). The mixture was treated as procedure A to afford
compound 2 (58.2 mg, 92.8%) as a yellow solid, and the properties
of compound 2 were listed as follows. IR (KBr): 1721, 1598
cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 3.81 (3H, s,
OCH.sub.3), 3.87 (3H, s, OCH.sub.3), 3.92 (3H, s, COOCH.sub.3),
6.95 (1H, d, J=8.8 Hz, H-3'), 7.05 (1H, dd, J=8.8, 3.2 Hz, H-4'),
7.21 (1H, d, J=3.2 Hz, H-6'), 7.51 (1H, d, J=16.0 Hz, H-.alpha.),
7.64 (2H, d, J=8.0 Hz, H-2 and 6), 7.65 (1H, d, J=16.0 Hz,
H-.beta.), 8.05 (2H, d, J=8.4 Hz, H-3 and 5). .sup.13C NMR
(CDCl.sub.3): .delta. 52.2 (COOCH.sub.3), 55.8 (OCH.sub.3), 56.4
(OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'), 119.6 (C-1' and 4'), 119.6
(C-.alpha.), 128.1 (C-2 and 6), 128.9 (C-3 and 5), 130.0 (C-4),
139.5 (C-1), 141.2 (C-.beta.), 152.8 (C-5'), 153.6 (C-2'), 166.5
(COOCH.sub.3), 191.8 (C.dbd.O). ESIMS: m/z 327 [M+H].sup.+. Anal.
Calcd for C.sub.19H.sub.18O.sub.5: C, 69.93; H, 5.56. Found: C,
69.95; H, 5.62.
[0034] 4. Preparation of compound 3
(4-ethoxycarbonyl-2',5'-dimethoxychalcone)
[0035] To a solution of compound 1 (100 mg, 0.32 mmol) in acetone
was added K.sub.2CO.sub.3 (88.5 mg, 0.64 mmol) and ethyl iodide
(0.64 mmol). The mixture was treated as procedure A to afford
compound 3 (50.0 mg, 45.9%) as a yellow solid, and the properties
of compound 3 were listed as follows. IR (KBr): 1715, 1599
cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 1.34 (3H, t, J=7.2 Hz,
CH.sub.3), 3.81 (3H, s, OCH.sub.3), 3.87 (3H, s, OCH.sub.3), 4.38
(2H, q, J=7.2 Hz, COOCH.sub.2--), 6.95 (1H, d, J=8.8 Hz, H-3'),
7.05 (1H, dd, J=8.8, 3.2 Hz, H-4'), 7.21 (1H, d, J=3.2 Hz, H-6'),
7.51 (1H, d, J=16.0 Hz, H-.alpha.), 7.64 (2H, d, J=8.4 Hz, H-2 and
6), 7.65 (1H, d, J=16.0 Hz, H-.beta.), 8.06 (2H, dd, J=8.4, 1.6 Hz,
H-3 and 5). .sup.13C NMR (CDCl.sub.3): .delta. 14.3 (CH.sub.3),
55.8 (OCH.sub.3), 56.4 (OCH.sub.3), 61.1 (COOCH.sub.2--), 113.4
(C-6'), 114.4 (C-3'), 119.6 (C-1' and 4'), 119.6 (C-.alpha.), 128.1
(C-2 and 6), 128.8 (C-3 and 5), 130.0 (C-4), 139.4 (C-1), 141.3
(C-.beta.), 152.8 (C-5'), 153.6 (C-2'), 166.0 (COOCH.sub.2--),
191.9 (C.dbd.O). ESIMS: m/z 341 [M+H].sup.+. Anal. Calcd for
C.sub.20H.sub.20O.sub.5.1/2H.sub.2O: C, 68.75; H, 6.06. Found: C,
68.71; H, 6.49.
[0036] 5. Preparation of compound 4
(propoxycarbonyl-2',5'-dimethoxychalcone)
[0037] To a solution of compound 1 (60 mg, 0.19 mmol) in acetone
was added K.sub.2CO.sub.3 (52.5 mg, 0.38 mmol) and propyl iodide
(0.38 mmol). The mixture was treated as procedure A to afford
compound 4 (27.8 mg, 40.8%) as a yellow oil, and the properties of
compound 4 were listed as follows. IR (KBr): 1715, 1602 cm.sup.-1.
.sup.1H NMR (CDCl.sub.3): .delta. 1.04 (3H, t, J=7.2 Hz, CH.sub.3),
1.80 (2H, m, CH.sub.2CH.sub.3), 3.81 (3H, s, OCH.sub.3), 3.88 (3H,
s, OCH.sub.3), 4.29 (2H, t, J=6.4 Hz, COOCH.sub.2--), 6.95 (11.1,
d, J=9.2 Hz, H-3'), 7.05 (1H, dd, J=9.2, 3.2 Hz, H-4'), 7.22 (1H,
d, J=3.2 Hz, H-6'), 7.51 (1H, d, J=16.0 Hz, H-.alpha.), 7.65 (2H,
d, J=8.4 Hz, H-2 and 6), 7.66 (1H, d, J=16.0 Hz, H-.beta.), 8.06
(2H, d, J=8.4 Hz, H-3 and 5). .sup.13C NMR (CDCl.sub.3): .delta.
10.5 (CH.sub.3), 22.1 (CH.sub.2CH.sub.3), 55.8 (OCH.sub.3), 56.4
(OCH.sub.3), 66.7 (COOCH.sub.2--), 113.4 (C-6'), 114.4 (C-3'),
119.7 (C-1' and 4'), 119.7 (C-.alpha.), 128.1 (C-2 and 6), 128.8
(C-3 and 5), 130.0 (C-4), 139.4 (C-1), 141.3 (C-.beta.), 152.8
(C-5'), 153.7 (C-2'), 166.1 (COOCH.sub.2--), 191.9 (C.dbd.O).
ESIMS: m/z 355 [M+H].sup.+. Anal. Calcd for
C.sub.21H.sub.22O.sub.5: C, 71.17; H, 6.26. Found: C, 71.15; H,
6.39.
[0038] 6. Preparation of compound 5
(4-isopropoxycarbonyl-2',5'-dimethoxychalcone)
[0039] To a solution of compound 1 (60 mg, 0.19 mmol) in acetone
was added K.sub.2CO.sub.3 (52.5 mg, 0.38 mmol) and isopropyl iodide
(0.38 mmol). The mixture was treated as procedure A to afford
compound 5 (58.2 mg, 85.6%) as a yellow oil, and the properties of
compound 5 were listed as follows. IR (KBr): 1711, 1603 cm.sup.-1.
.sup.1H NMR (CDCl.sub.3): .delta. 1.37 (6H, d, J=6.0 Hz,
CH.sub.3.times.2), 3.81 (3H, s, OCH.sub.3), 3.87 (3H, s,
OCH.sub.3), 5.26 (1H, m, COOCH(CH.sub.3).sub.2), 6.95 (1H, d, J=9.2
Hz, H-3'), 7.05 (1H, dd, J=8.8, 3.2 Hz, H-4'), 7.21 (1H, d, J=3.2
Hz, H-6'), 7.51 (1H, d, J=16.0 Hz, H-.alpha.), 7.64 (2H, d, J=8.8
Hz, H-2 and 6), 7.66 (1H, d, J=16.0 Hz, H-.beta.), 8.05 (2H, d,
J=8.4 Hz, H-3 and 5). .sup.13C NMR (CDCl.sub.3): .delta. 21.9
(CH.sub.3.times.2), 55.9 (OCH.sub.3), 56.5 (OCH.sub.3), 68.6
(COOCH(CH.sub.3).sub.2), 113.4 (C-6'), 114.4 (C-3'), 119.7 (C-1'
and 4'), 119.7 (C-.alpha.), 128.1 (C-2 and 6), 128.8 (C-3 and 5),
130.0 (C-4), 139.3 (C-1), 141.4 (C-.beta.), 152.8 (C-5'), 153.7
(C-2'), 166.1 (COOCH(CH.sub.3).sub.2), 191.9 (C.dbd.O). ESIMS: m/z
355 [M+H].sup.+. Anal. Calcd for
C.sub.21H.sub.22O.sub.5.1/4H.sub.2O: C, 70.28; H, 6.32. Found: C,
70.64; H, 6.26.
[0040] 7. Preparation of compound 6
(4-benzyloxycarbonyl-2',5'-dimethoxychalcone)
[0041] To a solution of compound 1 (60 mg, 0.19 mmol) in acetone
was added K.sub.2CO.sub.3 (52.5 mg, 0.38 mmol) and benzyl bromide
(0.38 mmol). The mixture was treated as procedure A to afford
compound 6 (67.7 mg, 87.6%) as a yellow oil, and the properties of
compound 6 were listed as follows. IR (KBr): 1717, 1601 cm.sup.-1.
.sup.1H NMR (CDCl.sub.3): .delta. 3.81 (3H, s, OCH.sub.3), 3.87
(3H, s, OCH.sub.3), 5.38 (2H, s, COOCH.sub.2--), 6.95 (1H, d, J=8.8
Hz, H-3'), 7.05 (1H, dd, J=8.8, 3.2 Hz, H-4'), 7.22 (1H, d, J=3.2
Hz, H-6'), 7.35 (5H, m, --C.sub.6H.sub.5), 7.51 (1H, d, J=16.0 Hz,
H-.alpha.), 7.64 (2H, d, J=8.4 Hz, H-2 and 6), 7.66 (1H, d, J=16.0
Hz, HA, 8.09 (2H, d, J=8.4 Hz, H-3 and 5). .sup.13C NMR
(CDCl.sub.3): .delta. 55.9 (OCH.sub.3), 56.5 (OCH.sub.3), 66.9
(COOCH.sub.2--), 113.4 (C-6'), 114.4 (C-3'), 119.7 (C-1' and 4'),
119.7 (C-.alpha.), 128.2-128.3 (tertiary aromatic carbon.times.5),
128.6 (C-2 and 6), 129.0 (C-3 and 5), 130.2 (C-4), 135.9
(quaternary aromatic carbon.times.1), 139.6 (C-1), 141.2
(C-.beta.), 152.8 (C-5'), 153.7 (C-2'), 165.8 (COOCH.sub.2--),
191.8 (C.dbd.O). ESIMS: m/z 403 [M+H].sup.+. Anal. Calcd for
C.sub.25H.sub.22O.sub.5: C, 74.61; H, 5.51. Found: C, 74.63; H,
5.63.
[0042] 8. Preparation of compound 7
(4-(3-methyl-but-2-enyloxycarbonyl)-2',5'-dimethoxychalcone)
[0043] To a solution of compound 1 (60 mg, 0.19 mmol) in acetone
was added K.sub.2CO.sub.3 (52.5 mg, 0.38 mmol) and
1-bromo-3-methyl-2-butene (0.38 mmol). The mixture was treated as
procedure A to afford compound 7 (62.5 mg, 85.5%) as a yellow oil,
and the properties of compound 7 were listed as follows. IR (KBr):
1714, 1600 cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 1.78 (3H,
s, CH.sub.3), 1.79 (3H, s, CH.sub.3), 3.81 (3H, s, OCH.sub.3), 3.88
(3H, s, OCH.sub.3), 4.83 (2H, d, J=7.2 Hz, COOCH.sub.2--), 5.47
(1H, m, CH.dbd.C(CH.sub.3).sub.2), 6.95 (1H, d, J=8.8 Hz, H-3'),
7.05 (1H, dd, J=8.8, 3.2 Hz, H-4'), 7.21 (1H, d, J=3.2 Hz, H-6'),
7.51 (1H, d, J=16.0 Hz, H-.alpha.), 7.63 (2H, d, J=8.0 Hz, H-2 and
6), 7.65 (1H, d, J=16.0 Hz, H-.beta.), 8.06 (2H, d, J=8.4 Hz, H-3
and 5). .sup.13C NMR (CDCl.sub.3): .delta. 18.1 (CH.sub.3), 25.8
(CH.sub.3), 55.9 (OCH.sub.3), 56.5 (OCH.sub.3), 62.1
(COOCH.sub.2--), 113.4 (C-6'), 114.4 (C-3'), 118.5
(CH.dbd.C(CH.sub.3).sub.2), 119.7 (C-1' and 4'), 119.7 (C-.alpha.),
128.1 (C-2 and 6), 128.8 (C-3 and 5), 130.1 (C-4), 137.1
(CH.dbd.C(CH.sub.3).sub.2), 139.4 (C-1), 141.4 (C-.beta.), 152.8
(C-5'), 153.7 (C-2'), 166.1 (COOCH.sub.2--), 191.9 (C.dbd.O).
ESIMS: m/z 381 [M+H].sup.+. Anal. Calcd for
C.sub.23H.sub.24O.sub.5: C, 72.61; H, 6.36. Found: C, 72.60; H,
6.48.
[0044] 9. Procedure B for synthesis of C-4 amide compounds 8 to
17
[0045] A mixture of compound 1 (156 mg, 0.5 mmol),
hydroxybenzotriazole (HOBt, 135.1 mg, 1.0 mmol) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl,
191.7 mg, 1.0 mmol) was dissolved with CH.sub.2Cl.sub.2, and
stirred for 10 minutes. The mixture was added amine (1.0 mmol), and
stirred at room temperature for 2 hours. The reaction mixture was
concentrated in vacuo to give the crude product. The crude product
was purified by chromatography with EtOAc/n-hexane, and
crystallized with EtOAc to afford purified products. Compounds 8 to
17 have the structural formula (Formula I) as follows, wherein R1
and R2 substituted groups are depicted in Table 1.
##STR00005##
[0046] The detailed descriptions for compounds 8 to 17 were
provided as follows.
[0047] 10. Preparation of compound 8
(4-methylcarbamoyl-2',5'-dimethoxychalcone)
[0048] A solution of compound 1 (156 mg, 0.5 mmol), HOBt (135.1 mg,
1.0 mmol) and EDCl (191.7 mg, 1.0 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
methylamine (1.0 mmol) and treated as procedure B to afford
compound 8 (82.3 mg, 50.6%) as a yellow solid, and the properties
of compound 8 were listed as follows. IR (KBr): 3326, 1652, 1546
cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 3.10 (3H, d, J=4.8 Hz,
NHCH.sub.3), 3.80 (3H, s, OCH.sub.3), 3.86 (3H, s, OCH.sub.3), 6.39
(1H, br s, CONH--), 6.94 (1H, d, J=9.2 Hz, H-3'), 7.04 (1H, dd,
J=9.2, 3.2 Hz, H-4'), 7.19 (1H, d, J=3.2 Hz, H-6'), 7.47 (1H, d,
J=16.0 Hz, H-.alpha.), 7.61 (2H, d, J=8.4 Hz, H-2 and 6), 7.62 (1H,
d, J=16.0 Hz, H-.beta.), 7.78 (2H, d, J=8.4 Hz, H-3 and 5).
.sup.13C NMR (CDCl.sub.3): .delta. 26.9 (NHCH.sub.3), 55.8
(OCH.sub.3), 56.5 (OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'), 119.5
(C-1' and 4'), 119.5 (C-.alpha.), 127.4 (C-2 and 6), 128.4 (C-3 and
5), 135.7 (C-4), 138.0 (C-1), 147.5 (C-.beta.), 152.7 (C-5'), 153.6
(C-2'), 167.5 (CONH--), 192.0 (C.dbd.O). ESIMS: m/z 326
[M+H].sup.+. Anal. Calcd for C.sub.19H.sub.19NO.sub.4: C, 70.14; H,
5.89; N, 4.31. Found: C, 69.23; H, 5.95; N, 4.21.
[0049] 11. Preparation of compound 9
(4-ethylcarbamoyl-2',5'-dimethoxychalcone)
[0050] A mixture of compound 1 (156 mg, 0.5 mmol), HOBt (135.1 mg,
1.0 mmol) and EDCl (191.7 mg, 1.0 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
ethylamine (1.0 mmol) and treated as procedure B to afford compound
9 (81.0 mg, 47.8%) as a yellow solid, and the properties of
compound 9 were listed as follows. IR (KBr): 3326, 1641, 1544
cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 1.25 (3H, t, J=7.2 Hz,
CH.sub.3), 3.50 (2H, m, NHCH.sub.2--), 3.80 (3H, s, OCH.sub.3),
3.86 (3H, s, OCH.sub.3), 6.26 (1H, br s, CONH--), 6.94 (1H, d,
J=8.8 Hz, H-3'), 7.04 (1H, dd, J=8.8, 3.2 Hz, H-4'), 7.20 (1H, d,
J=3.2 Hz, H-6'), 7.47 (1H, d, J=16.0 Hz, H-.alpha.), 7.62 (2H, d,
J=8.4 Hz, H-2 and 6), 7.63 (1H, d, J=16.0 Hz, H-.beta.), 7.78 (2H,
d, J=8.0 Hz, H-3 and 5). .sup.13C NMR (CDCl.sub.3): .delta. 14.8
(CH.sub.3), 35.0 (NHCH.sub.2--), 55.8 (OCH.sub.3), 56.5
(OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'), 119.5 (C-1' and 4'), 119.5
(C-.alpha.), 127.4 (C-2 and 6), 128.4 (C-3 and 5), 135.9 (C-4),
138.0 (C-1), 141.5 (C-.beta.), 152.7 (C-5'), 153.6 (C-2'), 166.7
(CONH--), 192.0 (C.dbd.O). EIMS (70 eV) m/z (% rel. int.): 339
(100). Anal. Calcd for C.sub.20H.sub.21NO.sub.4: C, 70.48; H, 6.24;
N, 4.13. Found: C, 69.87; H, 6.28; N, 4.07.
[0051] 12. Preparation of compound 10
(4-propylcarbamoyl-2',5'-dimethoxychalcone)
[0052] A mixture of compound 1 (156 mg, 0.5 mmol), HOBt (135.1 mg,
1.0 mmol) and EDCl (191.7 mg, 1.0 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
propylamine (1.0 mmol) and treated as procedure B to afford
compound 10 (52.9 mg, 30.0%) as a yellow oil, and the properties of
compound 10 were listed as follows. IR (KBr): 3329, 1641, 1544
cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 0.99 (3H, t, J=7.6 Hz,
CH.sub.3), 1.65 (2H, m, CH.sub.2CH.sub.3), 3.43 (2H, m,
NHCH.sub.2--), 3.81 (3H, s, OCH.sub.3), 3.87 (3H, s, OCH.sub.3),
6.22 (1H, br s, CONH--), 6.95 (1H, d, J=8.8 Hz, H-3'), 7.02 (1H,
dd, J=8.8, 3.2 Hz, H-4'), 7.20 (1H, d, J=3.2 Hz, H-6'), 7.48 (1H,
d, J=16.0 Hz, H-.alpha.), 7.63 (2H, d, J=8.8 Hz, H-2 and 6), 7.64
(1H, d, J=16.0 Hz, H-.beta.), 7.78 (2H, d, J=8.8 Hz, H-3 and 5).
.sup.13C NMR (CDCl.sub.3): .delta. 11.4 (CH.sub.3), 22.9
(CH.sub.2CH.sub.3), 41.8 (NCH.sub.2--), 55.9 (OCH.sub.3), 56.5
(OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'), 119.6 (C-1' and 4'), 119.6
(C-.alpha.), 127.4 (C-2 and 6), 128.4 (C-3 and C-5), 136.0 (C-4),
138.0 (C-1), 141.5 (C-.beta.), 152.7 (C-5'), 153.7 (C-2'), 166.8
(CONH--), 192.0 (C.dbd.O). ELMS (70 eV) m/z (% rel. int.): 353
(100). Anal. Calcd for C.sub.21H.sub.23NO.sub.4.1/2H.sub.2O: C,
69.58; H, 6.68; N, 3.87. Found: C, 70.60; H, 6.68; N, 3.85.
[0053] 13. Preparation of compound 11
(4-(2-methylethyl)carbamoyl-2',5'-dimethoxychalcone)
[0054] A mixture of compound 1 (100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol) and EDCl (122.7 mg, 0.64 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
isopropylamine (1.0 mmol) and treated as procedure B to afford
compound 11 (68.7 mg, 60.7%) as a yellow solid, and the properties
of compound 11 were listed as follows. IR (KBr): 3320, 1638, 1538
cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 1.26 (611, d, J=6.4
Hz, --CH(CH.sub.3).sub.2), 4.29 (1H, m, NHCH(CH.sub.3).sub.2), 6.02
(1H, d, J=7.6 Hz, CONH--), 6.94 (1H, d, J=8.8 Hz, H-3'), 7.04 (1H,
dd, J=9.0, 3.2 Hz, H-4'), 7.20 (1H, d, J=3.2 Hz, H-6'), 7.47 (1H,
d, J=16.0 Hz, H-.alpha.), 7.62 (2H, d, J=8.0 Hz, H-2 and 6), 7.63
(1H, d, J=16.0 Hz, H-.beta.), 7.77 (2H, d, J=8.4 Hz, H-3 and
5).
[0055] .sup.13C NMR (CDCl.sub.3): .delta. 22.8 (CH.sub.3), 29.7
(CH.sub.3), 42.0 (NHCH(CH.sub.3).sub.2), 55.8 (OCH.sub.3), 56.5
(OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'), 119.5 (C-1' and 4'), 119.5
(C-.alpha.), 127.4 (C-2 and 6), 128.4 (C-3 and 5), 136.1 (C-4),
138.0 (C-1), 141.5 (C-.beta.), 152.7 (C-5'), 153.6 (C-2'), 165.9
(CONH--), 192.0 (C.dbd.O). EIMS (70 eV) m/z (% rel. int.): 353
(100). Anal. Calcd for C.sub.20H.sub.23NO.sub.4: C, 71.37; H, 6.56;
N, 3.96. Found: C, 71.57; H, 6.68; N, 3.86.
[0056] 14. Preparation of compound 12
(4-dimethylcarbamoyl-2',5'-dimethoxychalcone)
[0057] A mixture of compound 1 (100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol) and EDCl (122.7 mg, 0.64 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
dimethylamine (1.0 mmol) and treated as procedure B to afford
compound 12 (53.0 mg, 48.8%) as a yellow oil, and the properties of
compound 12 were listed as follows. IR (KBr): 3485, 1633 cm.sup.-1.
.sup.1H NMR (CDCl.sub.3): .delta. 3.09 (6H, m,
--N(CH.sub.3).sub.2), 3.81 (3H, s, --OCH.sub.3), 3.86 (3H, s,
--OCH.sub.3), 6.94 (1H, d, J=9.2 Hz, H-3'), 7.03 (1H, dd, J=9.2,
3.2 Hz, H-4'), 7.19 (1H, d, J=3.2 Hz, H-6'), 7.44 (2H, d, J=8.4 Hz,
H-2 and 6), 7.45 (1H, d, J=16.0 Hz, H-.alpha.), 7.51 (2H, d, J=8.0
Hz, H-3 and 5), 7.63 (1H, d, J=16.0 Hz, H-.beta.). .sup.13C NMR
(CDCl.sub.3): .delta. 35.3 and 39.5 (N(CH.sub.3).sub.2), 55.8
(OCH.sub.3), 56.5 (OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'), 119.5
(C-1' and 4'), 119.5 (C-.alpha.), 127.6 (C-2 and 6), 128.3 (C-3 and
5), 136.3 (C-4), 137.7 (C-1), 141.8 (C-.beta.), 152.7 (C-5'), 153.6
(G-2'), 170.9 (CONH--), 192.0 (C.dbd.O). EIMS (70 eV) m/z (% rel.
int.): 339 (100). Anal. Calcd for C.sub.20H.sub.21NO.sub.4: C,
70.48; H, 6.24; N, 4.13. Found: C, 70.10; H, 6.45; N, 4.16.
[0058] 15. Preparation of compound 13
(4-cyclohexylcarbamoyl-2',5'-dimethoxychalcone)
[0059] A mixture of compound 1 (100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol) and EDCl (122.7 mg, 0.64 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
cyclohexylamine (1.0 mmol) and treated as procedure B to afford
compound 13 (84.0 mg, 66.7%), and the properties of compound 13
were listed as follows. IR (KBr): 3312, 1637, 1540 cm.sup.-1.
.sup.1H NMR (CDCl.sub.3): .delta. 1.24 (2H, m, --CH.sub.2--), 1.44
(2H, m, 1.66 (2H, m, --CH.sub.2--), 1.76 (2H, m, --CH.sub.2--),
2.04 (2H, m, --CH.sub.2--), 3.81 (3H, s, OCH.sub.3), 3.94 (3H, s,
OCH.sub.3), 3.98 (1H, m,
##STR00006##
6.00 (1H, d, J=7.6 Hz, CONH--), 6.95 (1H, d, J=8.8 Hz, H-3'), 7.05
(1H, dd, J=8.8, 3.2 Hz, H-4'), 7.21 (1H, d, J=3.2 Hz, H-6'), 7.48
(1H, d, J=16.0 Hz, H-.alpha.), 7.63 (2H, d, J=7.6 Hz, H-2 and 6),
7.64 (1H, d, J=16.0 Hz, H-.beta.), 7.77 (2H, d, J=8.4 Hz, H-3 and
5). .sup.13C NMR (CDCl.sub.3): .delta. 24.9 (--CH.sub.2--.times.2),
25.5 (--CH.sub.2--.times.1), 33.2 (--CH.sub.2--.times.2), 48.8
##STR00007##
55.9 (OCH.sub.3), 56.5 (OCH.sub.3), 113.4 (C-6'), 114.4 (C-3'),
119.6 (C-1' and 4'), 119.6 (C-.alpha.), 127.4 (C-2 and 6), 128.4
(C-3 and 5), 136.2 (C-4), 138.0 (C-1), 141.5 (C-.beta.), 152.7
(C-5'), 153.7 (C-2'), 165.8 (CONH--), 192.0 (C.dbd.O) EIMS (70 eV)
m/z (% rel. int.): 393 (100). Anal. Calcd for
C.sub.24H.sub.27NO.sub.4: C, 73.26; H, 6.92; N, 3.56. Found: C,
73.31; H, 7.03; N, 3.55.
[0060] 16. Preparation of compound 14
(4-diethylcarbamoyl-2',5'-dimethoxychalcone)
[0061] A mixture of compound 1 (100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol) and EDCl (122.7 mg, 0.64 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
diethylamine (1.0 mmol) as procedure B to afford compound 14 (115.5
mg, 98.2%) as a yellow oil, and the properties of compound 14 were
listed as follows. IR (KBr): 3568, 1627 cm.sup.-1. .sup.1H NMR
(CDCl.sub.3): .delta. 1.12 (3H, m, CH.sub.3), 1.24 (3H, m,
CH.sub.3), 3.25 (2H, m, NCH.sub.2--), 3.54 (2H, m, NCH.sub.2--),
3.80 (3H, s, OCH.sub.3), 3.86 (3H, s, OCH.sub.3), 6.94 (1H, d,
J=8.8 Hz, H-3'), 7.03 (1H, dd, J=9.2, 3.2 Hz, H-4'), 7.19 (1H, d,
J=2.8 Hz, H-6'), 7.39 (2H, d, J=8.4 Hz, H-2 and 6), 7.44 (1H, d,
J=15.6 Hz, H-.alpha.), 7.60 (2H, d, J=8.0 Hz, H-3 and H-5), 7.63
(1H, d, J=15.6 Hz, H-fi).
[0062] .sup.13C NMR (CDCl.sub.3): .delta. 12.9 (CH.sub.3), 14.2
(CH.sub.3), 39.3 (NCH.sub.2--), 43.2 (NCH.sub.2--), 55.8
(OCH.sub.3), 56.4 (OCH.sub.3), 113.3 (C-6'), 114.4 (C-3'), 119.4
(C-1' and 4'), 119.4 (C-.alpha.), 126.9 (C-2 and 6), 128.4 (C-3 and
5), 135.9 (C-4), 138.7 (C-1), 141.9 (C-.beta.), 152.6 (C-5'), 153.6
(C-2'), 170.5 (--CONH--), 192.1 (C.dbd.O). ELMS (70 eV) m/z (% rel.
int.): 367 (100). Anal. Calcd for
C.sub.22H.sub.25NO.sub.4.1/4H.sub.2O: C, 71.04; H, 6.91; N, 3.77.
Found: C, 71.23; H, 6.93; N, 3.65.
[0063] 17. Preparation of compound 15
(4-tetrahydropyrrolylcarbamoyl-2',5'-dimethoxychalcone)
[0064] A mixture of compound 1 (100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol) and EDCl (122.7 mg, 0.64 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
pyrrolidine (1.0 mmol) and treated as procedure B to afford
compound 15 (80.0 mg, 68.4%) as a yellow oil, and the properties of
compound 15 were listed as follows. IR (KBr): 3466, 1619 cm.sup.-1.
.sup.1H NMR (CDCl.sub.3): .delta. 1.89 (4H, m,
--CH.sub.2CH.sub.2--), 3.42 (2H, m, NCH.sub.2--), 3.60 (2H, m,
NCH.sub.2--), 3.79 (3H, s, OCH.sub.3), 3.85 (3H, s, OCH.sub.3),
6.94 (1H, d, J=8.8 Hz, H-3'), 7.02 (1H, dd, J=8.0, 3.2 Hz, H-4'),
7.18 (1H, d, J=3.2 Hz, H-6'), 7.44 (1H, d, J=16.0 Hz, H-.alpha.),
7.52 (2H, d, J=8.0 Hz, H-2 and 6), 7.59 (2H, d, J=8.4 Hz, H-3 and
5), 7.62 (1H, d, J=16.0 Hz, H-.beta.). .sup.13C NMR (CDCl.sub.3):
.delta. 24.3 (--CH.sub.2CH.sub.2--), 26.3 (--CH.sub.2CH.sub.2--),
46.2 (NCH.sub.2--), 49.5 (NCH.sub.2--), 55.8 (OCH.sub.3), 56.4
(OCH.sub.3), 113.3 (C-6'), 114.3 (C-3'), 119.4 (C-1' and 4'), 119.4
(C-.alpha.), 127.6 (C-2 and 6), 128.1 (C-3 and 5), 136.5 (C-4),
138.5 (C-1), 141.8 (C-.beta.), 152.6 (C-5'), 153.5 (C-2'), 168.9
(CONH--), 192.0 (C.dbd.O). EIMS (70 eV) m/z (% rel. int.): 365
(97), 295 (100). Anal. Calcd for
C.sub.22H.sub.23NO.sub.4.1/4H.sub.2O: C, 71.43; H, 6.40; N, 3.79.
Found: C, 71.67; H, 6.71; N, 3.6.3
[0065] 18. Preparation of compound 16
(4-(2-hydroxyethyl)methyl-carbamoyl-2',5'-dimethoxychalcone)
[0066] A mixture of compound 1 (100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol) and EDCl (122.7 mg, 0.64 mmol) was dissolved with
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
N-methylethanolamine (1.0 mmol) and treated as procedure B to
afford compound 16 (93.6 mg, 79.1%) as a yellow oil, and the
properties of compound 16 were listed as follows. IR (KBr): 3403,
1610 cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 2.45 (OH), 3.05
(3H, s, NCH.sub.3), 3.10 (1H, m, NCHH--), 3.42 (1H, m, NCHH--),
3.71 (2H, m, CH.sub.2OH), 3.80 (3H, s, OCH.sub.3), 3.85 (3H, s,
OCH.sub.3), 6.93 (1H, d, J=9.2 Hz, H-3'), 7.03 (1H, dd, J=8.8, 3.2
Hz, H-4'), 7.19 (1H, d, J=3.2 Hz, H-6'), 7.45 (1H, d, J=16.0 Hz,
H-.alpha.), 7.46 (2H, d, J=8.0 Hz, H-2 and 6), 7.61 (1H, d, J=9.2
Hz, H-3 and 5), 7.62 (1H, d, J=16.0 Hz, H-.beta.). .sup.13C NMR
(CDCl.sub.3): .delta. 38.6 (NCH.sub.3), 51.0 (NCH.sub.2--), 55.8
(OCH.sub.3), 56.4 (OCH.sub.3), 60.6 (--CH.sub.2OH), 113.3 (C-6'),
114.4 (C-3'), 119.5 (C-1' and 4'), 119.5 (C-.alpha.), 127.8 (C-2
and 6), 128.3 (C-3 and 5), 136.7 (C-4), 137.0 (C-1), 141.6
(C-.beta.), 152.7 (C-5'), 153.6 (C-2'), 172.5 (--CON--), 192.0
(C.dbd.O). EIMS (70 eV) m/z (% rel. int.): 369 (31), 295 (100).
Anal. Calcd for C.sub.21H.sub.23NO.sub.5: C, 68.28; H, 6.28; N,
3.79. Found: C, 66.78; H, 6.52; N, 3.45.
[0067] 19. Preparation of compound 17
(N-cyclopropyl-4-[3-cyclopropylimino-3-(2',5'-dimethoxy-phenyl)-propenyl]-
-benzamide)
[0068] A mixture of compound 1 (100 mg, 0.32 mmol), HOBt (86.5 mg,
0.64 mmol) and EDCl (122.7 mg, 0.64 mmol) was dissolved in
CH.sub.2Cl.sub.2, and stirred for 10 minutes. The mixture was added
cyclopropylamine (1.0 mmol) and treated as procedure B to afford
compound 17 (95.3 mg, 76.2%) as a yellow solid, and the properties
of compound 17 were listed as follows. IR (KBr): 3286, 1640, 1536
cm.sup.-1. .sup.1H NMR (CDCl.sub.3): .delta. 0.60 (2H, m,
--CH.sub.2CH.sub.2--), 0.84 (4H, m, --CH.sub.2CH.sub.2), 0.96 (2H,
m, --CH.sub.2CH.sub.2--), 2.67 (1H, m, NHCH--), 2.87 (1H, m,
.dbd.NHCH--), 6.28 (1H, br s, CONH--), 6.42 (1H, d, J=16.0 Hz,
H-.alpha.), 6.70 (1H, d, J=2.4 Hz, H-6'), 6.95 (2H, m, H-3' and
4'), 7.12 (1H, d, J=16.0 Hz, H-.beta.), 7.42 (2H, d, J=8.4 Hz, H-2
and 6), 7.66 (2H, d, J=8.4 Hz, H-3 and H-5). .sup.13C NMR
(CDCl.sub.3): .delta. 6.8 (--CH.sub.2--.times.2), 9.9
(--CH.sub.2--), 10.0 (--CH.sub.2--), 23.1 (--NHCH--), 36.2
(.dbd.NCH--), 55.8 (OCH.sub.3), 56.4 (OCH.sub.3), 112.6 (C-6'),
114.5 (C-3'), 115.0 (C-1' and 4'), 125.7 (C-.alpha.), 127.1 (C-2),
133.5 (C-5 and C-6), 133.7 (C-3), 135.6 (C-1), 139.6 (C-.beta.),
150.5 (C-5'), 153.6 (C-2'), 164.6 (C.dbd.N--), 168.3 (CONH--). EIMS
(70 eV) m/z (% rel. int.): 390 (31). Anal. Calcd for
C.sub.24H.sub.26N.sub.2O.sub.3.1/4H.sub.2O: C, 72.98; H, 6.76; N,
7.09. Found: C, 73.01; H, 6.69; N, 7.10.
[0069] 20. Preparation of compound 18
(3-(3-thiophene)carboxyl-1-(2,5-dimethoxyphenyl)prop-2-en-1-one)
[0070] 2,5-Dimethoxyacetophenone (450.0 mg, 2.5 mmol) and
5-formyl-2-thiophenecarboxylic acid (390.4 mg, 2.5 mmol) were
dissolved in MeOH (50 mL), and added 8% KOH in H.sub.2O (50 mL).
The reaction mixture was stirred at room temperature for 72 hours
and neutralized with 10% HCl solution (100 mL) to form yellow
precipitate. The yellow precipitate was filtered and washed with
appropriate amount of water. The crude product was purified by
chromatography using EtOAc/n-hexane (2:1), and crystallized with
EtOAc to afford compound 18 (469.6 mg, 59.1%) as a yellow solid.
The reaction formula of compound 18 (Formula II) was listed as
follows.
##STR00008##
[0071] The properties of compound 18 were listed as follows.
.sup.1H NMR (CD.sub.3OD): .delta. 3.80 (3H, s, OCH.sub.3), 3.90
(3H, s, OCH.sub.3), 7.12 (1H, m, H-3'), 7.14 (1H, m, H-4'), 7.15
(1H, m, H-6'), 7.40 (1H, d, J=15.6 Hz, H-.alpha.), 7.42 (1H, d,
J=3.2 Hz, H-5), 7.70 (1H, d, 15.6 Hz, H-.beta.), 7.71 (1H, d, J=3.2
Hz, H-4). .sup.13C NMR (CD.sub.3OD): .delta. 56.2 (OCH.sub.3), 56.9
(OCH.sub.3), 114.8 (C-6'), 115.5 (C-3'), 120.7 (C-4'), 129.1
(C-.alpha.), 132.8 (C-5), 135.1 (C-4), 135.8 (C-.beta.), 147.5
(C-1), 154.3 (C-5'), 155.2 (C-2'), 193.2 (C.dbd.O). ESIMS: m/z 319
[M+H].sup.+. Anal. Calcd for C.sub.16H.sub.14O.sub.5S: C, 60.37; H,
4.43; S, 10.07. Found: C, 60.09; H, 4.42; S, 10.27.
[0072] II. Cell Viability/Proliferation Experiments
[0073] The cell lines of the present invention were an human
urothelial carcinoma cell line (NTUB1), a human prostate cancer
cell line (PC3), a human lung adenocarcinoma epithelial cell line
(A549) and a SV-40 immortalized human uroepithelial cell line
(SV-HUC1). However, the cell lines applied in the present invention
includes but not limit in NTUB1, PC3, A549 and SV-HUC1 cells. Other
cell lines having the same characteristics or not exactly the same
characteristics are able to be used in the present invention.
NTUB1, PC3 and A549 cells were maintained in RPMI 1640 and SV-HUC1
cells were maintained in F12 medium supplemented with 10% fetal
bovine serum (FBS), 100 unit/mL penicillin-G, 100 .mu.g/mL
streptomycin and 2 mM glutamine. The cells were cultured at
37.degree. C. in a humidified atmosphere containing 5% carbon
dioxide.
[0074] The cytotoxic effect of compounds 1 to 18 of the present
invention and cisplatin (positive control) on the cell lines was
evaluated using
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay. Briefly, the cells were plated at a density of 1800
cells/well in 96-well plates and incubated in the presence of
graded concentrations of compounds 1 to 18 and cisplatin at
37.degree. C. for 72 hours. At the end of the culture period, 50
.mu.L of MTT (2 .mu.g mg/mL in PBS) was added to each well and
allowed to react for 3 hours. Following centrifugation of plates at
1000 g for 10 minutes, media were removed and 150 .mu.L dimethyl
sulfoxide (DMSO) were added to each well. The proportions of
surviving cells were determined by absorbance spectrometry at 540
nm. The cell viability was expressed as a percentage to the viable
cells of control culture condition. The IC.sub.50 value of each
group were calculated by the median-effect analysis and presented
as mean.+-.standard deviation (SD).
[0075] III. Cell Cycle Analysis
[0076] DNA content was determined following propidium iodide (PI)
staining of cells. Briefly, 6.times.10.sup.5 cells were plated and
treated with 20 .mu.M cisplatin and various concentrations of
compounds 11 and 15 for 24 hours, respectively. These cells were
harvested by trypsinization, washed with 1.times.PBS, and fixed in
ice-cold MeOH at -20.degree. C. After overnight incubation, the
cells were washed with PBS and incubated with 50 .mu.g/mL propidium
iodide and 50 .mu.g/mL RNase A in PBS at room temperature for 30
minutes. The fractions of cells in each phase of cell cycle were
analyzed using flow cytometer.
[0077] IV. Immuno-Fluorescence Staining
[0078] In brief, NTUB 1 cells were seeded onto serum-coated
coverslides. The cells were fixed with 2% formaldehyde/PBS for 20
minutes followed by cold methanol permeabilization for 3 minutes.
The cells were washed with 1.times.PBS five minutes twice. The
cells were then incubated with .alpha.-tubulin primary antibody
overnight. The cells were washed with 1.times.PBS five minutes
thrice. Goat anti-rabbit-rhodamine secondary antibody and 1
.mu.g/mL 4'-6-diamidino-2-phenylindole (DAPI) were incubated for 1
hour at room temperature. The slides were washed with 1.times.PBS,
mounted with mounting solution (80% glycerol/PBS) and photographed
by microscopy.
[0079] V. Western Blot Analysis for .alpha.-Tubulin
[0080] NTUB1 cells were seeded at a density of
1.times.10.sup.6/100-mm.sup.2 dishes and treated with indicated
concentrations of test agents for 6 hours. The cells were then
washed with cold PBS and lysed by adding lysis buffer (20 mM
Tris-HCl (pH 6.8), 1 mM MgCl.sub.2, 2 mM EGTA (ethylene glycol
tetraacetic acid), 20 g/mL aprotinin, 20 g/mL leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 1 mM orthovanadate, and 0.5% Nonidet
P-40). The cell lysates were centrifuged at 15,000 g for 10 min at
4.degree. C. Soluble and assembled tubulin could be separated in
supernatant and pellet fraction, respectively. The supernatants and
pellets were revolutionized by the sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis assay. The relative
amounts of tubulins transferred on the nitrocellulose membrane were
detected by anti-tubulin monoclonal antibody and horseradish
peroxidase (HRP)-conjugated secondary antibody. .beta.-Actin was
used for control. Detection of immunoreactive signal was
accomplished with Western blot chemiluminescent reagent.
EXPERIMENTAL RESULTS
[0081] Please refer to Table 2, which depicts that compound 1 and
most of its derivatives showed significantly cytotoxic activities
against NTUB1 cells while compound 18 had no significant
cytotoxicity against NTUB1 cells. The esterification of C-4-COOH of
compound 1 enhanced the cytotoxicity against NTUB1 and PC3 cells.
The amidation of C-4-COOH of compound 1 enhanced he cytotoxic
effect against the two cancer cell lines while compounds 10, 11 and
15 significantly enhanced the cytotoxicity against NTUB1 and PC3
cells. In Table 2, the compounds possessed of 4-propyl,
4-(2-methylethyl) and 4-tetrahydropyrrolyl carbamoyl groups
significantly enhanced the cytotoxicity against NTUB1 and PC3 cell
lines.
[0082] Please refer to FIG. 1, compound 15 showed more potent
cytotoxic effect against NTUB1 and PC3 cells than that of A549
cells. It indicated that compound 15 displayed the specific
cytotoxicity against cancer cells in the urinary system. In
addition, compound 15 exhibited less cytotoxicity against human
normal uroepithelial SV-HUC1 cells than those of NTUB1 and PC3
cells lines. It suggested that compound 15 was a good candidate for
developing as cancer chemotherapeutic agents for treating cancer
related to urinary system.
[0083] Next, the effect of positive control (cisplatin) and
compounds 11 and 15 on cell cycle progression of cancer cells was
determined, and the control was indicated to the absence of drug.
As shown in FIG. 2, NTUB1 cells treated with 20 .mu.M cisplatin for
24 hours led to an accumulation of cells in G1 and S phases with
the concomitant increase of the population of sub-G1 phase. NTUB1
cells were exposed to 1 .mu.M compounds 11 and 15, respectively,
for 24 hours induced G1 phase arrest compared to the respective
control value, accompanied by an increase in apoptotic cell death,
respectively. As shown in FIG. 3, PC3 cells treated with 20 .mu.M
cisplatin for 24 hours led to an accumulation of cells in S phase
with the concomitant increase of apoptotic cell death. Treatment of
PC3 cells with 1 .mu.M compounds 11 and 15, respectively, induced S
and G1 phase arrests, respectively, compared to respective control
value, accompanied by an increase of apoptotic cells while exposure
of PC3 cells to 0.3 .mu.M compounds 11 and 15, respectively,
induced G1 and G2/M phase arrests, respectively, compared to the
respective control value, accompanied by an increase of the
apoptotic cells death. The aforementioned results suggested that
compounds 11 and 15 at lower dose revealed same cell cycle arrest
in NTUB1 cells while their at higher dose indicated different cell
cycle arrest in PC3 cells.
[0084] In addition, NTUB1 cells treated with different
concentrations of compound 15 for 24 hours showed more cell death
and rounding up after treatment (data not shown). Microtubules play
an important role in the cell differentiation. If the function of
microtubules is blocked by chemicals or compounds (such as
paclitaxel), cell differentiation will arrest and be not progressed
into the normal pattern of the cell cycle control. It was known
from the immuno-fluorescent microscopy that compound 15 induced the
microtubule bundle formation in NTUB1 cells and mimicked the effect
of paclitaxel. NTUB1 cells treated with 1 .mu.M compound 15 showed
that high dosage of compound 15 induced more NTUB1 cells arrested
at mitosis and influenced microtubule formation (data not shown).
Following compound 15 binding to .alpha.-tubulin, it inhibited
microtubule dynamic instability, cell cycle G2/M phase transition,
mitotic arrest and NTUB1 cell death through apoptosis (as shown in
FIG. 2).
[0085] Please refer to FIG. 4, NTUB1 cells were treated with 500 nM
paclitaxel and more high different concentrations of compound 15
for 6 hours and collected the supernatant and pellet for assays.
Paclitaxel, used as the positive control, certainly stabilized the
tubulin assembly. .alpha.-Tubulin in the supernatant showed a
decreased level in a dose-dependent manner after treatment with
various concentrations of compound 15 while in the pellet increased
in a dose-dependent manner. It suggested that compound 15 could
influence tubulin assembly in the molecular action similar to
paclitaxel.
[0086] Therefore, in the present invention,
4-carboxyl-2',5'-dimethoxychalcone synthesized from
2,5-dimethoxyacetophenone and methyl 4-formylbenzoate and its
2',5'-dimethoxylchalcones showed the cytotoxicity on the urinary
system-related cancer cells NTUB1 and PC3, wherein compound 15 with
a carbamoyl group substituted at C-4 of B ring was found to have
strongest cytotoxicity against NTUB1 and PC3 cancer cell lines and
showed less cytotoxicity against SV-HUC1 and A549 cells. Thus, it
indicated compound 15 specifically inhibited the growth of cancer
cells in urinary system. It also indicated that the carbamoyl group
substituted at C-4 of the B ring of chalcone moiety may also
important structural feature for binding to tubulin. It suggested
that compound 15 may be used as microtubule-targeted
tubulin-polymerizing agents (MTPAs).
[0087] The R1 and R2 substituted groups of the synthesized
2',5'-dimethoxylchalcone derivatives in the present invention
includes but not limit in the substituted groups of compounds 1 to
17. One skilled in the art also can use the derivatives having the
substituted groups described in the present invention to synthesize
other 2',5'-dimethoxylchalcone derivatives.
[0088] While the invention has been described in terms of what is
presently considered to be the most practical and preferred
Embodiments, it is to be understood that the invention needs not be
limited to the disclosed Embodiments. On the contrary, it is
intended to cover various modifications and similar arrangements
included within the spirit and scope of the appended claims, which
are to be accorded with the broadest interpretation so as to
encompass all such modifications and similar structures.
TABLE-US-00001 TABLE 1 Side chains, melting points, yield and
molecular formulas of compounds 1 to 18 Compound R1 R2 Mp (.degree.
C.) Yield (%) Molecular formula 1 --OH O 214-215 63.0
C.sub.18H.sub.16O.sub.5 2 --OCH.sub.3 O 118-119 92.8
C.sub.19H.sub.18O.sub.5 3 --OCH.sub.2CH.sub.3 O 118-119 45.9
C.sub.20H.sub.20O.sub.5.cndot.1/2H.sub.2O 4
--OCH.sub.2CH.sub.2CH.sub.3 O Oil 40.8 C.sub.21H.sub.22O.sub.5 5
--OCH(CH.sub.3).sub.2 O Oil 85.6
C.sub.21H.sub.22O.sub.5.cndot.1/4H.sub.2O 6
--OCH.sub.2C.sub.6H.sub.5 O Oil 87.6 C.sub.25H.sub.22O.sub.5 7
--OCH.sub.2CH.dbd.C(CH.sub.3).sub.2 O Oil 85.5
C.sub.23H.sub.24O.sub.5 8 --NHCH.sub.3 O 217-218 50.6
C.sub.19H.sub.19NO.sub.4 9 --NHCH.sub.2CH.sub.3 O 213-214 47.8
C.sub.20H.sub.21NO.sub.4 10 --NHCH.sub.2CH.sub.2CH.sub.3 O Oil 30.0
C.sub.21H.sub.23NO.sub.4.cndot.1/2H.sub.2O 11
--NHCH(CH.sub.3).sub.2 O 227-228 60.7 C.sub.20H.sub.23NO.sub.4 12
--N(CH.sub.3).sub.2 O Oil 48.8 C.sub.20H.sub.21NO.sub.4 13
##STR00009## O 280-281 66.7 C.sub.24H.sub.27NO.sub.4 14
--N(CH.sub.2CH.sub.3).sub.2 O Oil 98.2
C.sub.22H.sub.25NO.sub.4.cndot.1/4H.sub.2O 15 ##STR00010## O Oil
68.4 C.sub.22H.sub.23NO.sub.4.cndot.1/4H.sub.2O 16 ##STR00011## O
Oil 79.1 C.sub.21H.sub.23NO.sub.5 17 ##STR00012## ##STR00013##
282-283 76.2 C.sub.24H.sub.26N.sub.2O.sub.3.cndot.1/4H.sub.2O 18
237-238 59.1 C.sub.16H.sub.14O.sub.5S
TABLE-US-00002 TABLE 2 Cytotoxicities of chalcone derivatives
against NTUB1 and PC3 cancer cell lines.sup.a NTUB1 PC3 Compound
IC.sub.50 (.mu.M) .+-. SD IC.sub.50 (.mu.M) .+-. SD Cisplatin 3.27
.+-. 0.10 4.56 .+-. 0.76 1 5.70 .+-. 0.68 7.24 .+-. 0.58 2 3.51
.+-. 0.45 4.85 .+-. 0.11 3 4.49 .+-. 0.98 5.70 .+-. 0.43 4 6.01
.+-. 0.30 26.93 .+-. 0.00 5 4.42 .+-. 0.50 6.74 .+-. 1.34 6 13.26
.+-. 2.86 17.95 .+-. 0.50 7 13.41 .+-. 0.78 >50 .mu.M 8 2.20
.+-. 0.37 2.13 .+-. 0.21 9 3.76 .+-. 0.40 4.18 .+-. 0.23 10 2.26
.+-. 0.31 1.50 .+-. 0.22 11 1.97 .+-. 0.39 1.58 .+-. 0.08 12 3.89
.+-. 0.21 4.58 .+-. 1.05 13 2.25 .+-. 0.57 6.30 .+-. 0.59 14 2.69
.+-. 0.47 3.79 .+-. 0.56 15 1.26 .+-. 0.25 0.53 .+-. 0.00 16 4.42
.+-. 0.15 5.75 .+-. 1.27 17 3.57 .+-. 0.47 4.26 .+-. 0.81 18 >50
.mu.M n.d .sup.aPositive control: cisplatin; n.d, not determined, n
= 3-5.
* * * * *